"NFκBp50-p65, P-TEFb and TNFα influence MYB elongation.(A) 293 cells were treated with the P-TEFb inhibitor DRB for 6 h and endogenous MYB expression assessed by QPCR. (B) Transactivation studies in 293 cells using 2 μg of the MYB SLR polyU CAT reporter and 0.125 μg of pcDNA NFκBp50-p65. At 12 h post transfection cells were treated with DRB and incubated for a further 24 h. (C) NFκBp50-p65 induces endogenous MYB. Total RNA was isolated from 293 cells transfected with; 1 μg of pcDNA NFκB p50-p65, 1 μg of pcDNA NFκBp50 K148A-p65 or 4 μg of pcDNA NFκBp50 K146-148A-p65 and analyzed by Q-PCR to measure MYB expression levels and (D) 1 μg of pcDNA NFκB p50-p65 and analyzed by Q-PCR to measure intronic pre-mRNA MYB transcript upstream (preSLR) and downstream (post SLR) of the MYB SLR polyU. Data are expressed as a ratio ""post/pre"", a measure of the amount of transcription through the SLR. (E) ChIP analysis of RNA polymerase II levels at the MYB SLR polyU. 293 cells were transfected with pcDNA, NFκB p50-p65 or pcDNA NFκBp50 K146-148A-p65. Cross-linked chromatin extracts were prepared at 48h post transfection and RNA polymerase II was detected by anti-pol II followed by Q-PCR. (F) SI organoids cultures and 293 cells were exposed to TNFα (20–100 ng/ml) for the times indicated and total RNA was isolated and analyzed by Q-PCR to measure MYB expression levels. (G) 293 cells were transfected with 2 μg of the MYB SLR polyU CAT or MYB ΔSLR polyU CAT reporter. At 24 h post transfection cells were exposed to 100 ng/ml TNFα for 10 h and CAT reporter activity assessed. (H) 293 cells were exposed to BAY inhibitor for the times indicated and total RNA was isolated and analyzed by Q-PCR to measure MYB expression levels. Error bars represent mean ± SEM, * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001."
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